UJMR, Volume 6 Number 1, June, 2021, pp 208 - 218 ISSN: 2616 - 0668

https://doi.org/10.47430/ujmr.2161.028

Received: 11th June, 2021 Accepted: 15th June, 2021

Determination of Aflatoxin Concentrations in Cereals and Legumes Marketed in

Zaria Metropolis, Kaduna State, Nigeria

Shitu, S.1*, Attahiru, M.1 and Umar, H.2

1
Department of Applied Biology, College of Science and Technology, Kaduna Polytechnic,
Kaduna State, Nigeria
2
Department of Applied Chemistry, College of Science and Technology, Kaduna
Polytechnic, Kaduna State, Nigeria
*Corresponding Author; Email: sabiushitu22@gmail.com
Abstract
Aflatoxins are group of secondary fungal metabolites produced by Aspergillus species, such as
Aspergillus flavus and Aspergillus parasiticus. The aflatoxin producing moulds can grow on cereals
and legumes in the field, poorly dried harvested crops in storage, processed food, and feed
products. The study was carried out with the aim to determine the level of aflatoxin
contamination of cereals grain and legumes in Zaria metropolis, Kaduna State, Nigeria. Ninety
(90) samples were collected, which comprises of 18 samples each of millet, sorghum, maize,
beans, and groundnuts respectively. The samples were subjected to proximate analysis. The
grains were further subjected to cultural isolation and microscopic identification. The isolates
were then screened for aflatoxin production ability with neutral red desiccated coconut agar and
viewed under UV light (365nm). The remaining portions of the samples was grounded and
extracted with 80% (v/v) methanol. The enzyme-linked Immunosorbent Assay (ELISA) technique
was used in quantifying the total aflatoxin content of the samples. The results revealed that all
the cereals and legumes analysed contain organic and inorganic nutrients that can support the
growth of aflatoxigenic moulds and production of aflatoxins. Some major parameters such as
carbohydrate content, crude protein, crude lipid, and ash contents were statistically significant
(p < 0.05). Thirty-one (31) isolates from the 90 samples were confirmed to be A. flavus and
seventeen (17) were A. Parasiticus, with percentage occurrence of 34.4% and 18.9% respectively.
All the isolates were screened and demonstrated ability for aflatoxin production under Ultra-
Violent light (390nm). The results also revealed a high concentration of aflatoxin (11.04 µg/kg) in
millet and a low concentration in sorghum (1.07 µg/kg). The contamination levels within the
grains were found to be statistically significant (p < 0.05). Aflatoxin contaminations also occurred
in 48 samples out of the 90 samples analysed. The grains samples analysed were found to be
contaminated with varying amounts of aflatoxins, which is harmful to humans and animals.
Therefore, steps should be taken to ensure that grains are properly dried prior to storage.
Keywords: aflatoxin, A. flavus, A. parasiticus, cereals, Enzyme-linked Immunosorbent Assay,
legumes

INTRODUCTION compliment the protein supplied by cereals.

Cereals are crop plants belonging to the grass
family Poaceae that are cultivated for their
edible starchy seeds or grains and botanically
known as caryopsis (Ukonmah and Eruotor,
2012). The term ‘cereal’ applies to the entire
plant as well as the grain and it is also loosely
applied to the grain product. The cereal grain is
a one seeded indehiscent fruit, or caryopsis, in
which the pericarp is completely fused with the
seed coat (Ukonmah and Eruotor, 2012).
Legumes belong to the family Fabaceae
(Leguminosae). Legumes are second only to
cereals as source of human food and provide
much needed proteins. Legumes contain
relatively high amounts of essential amino
acids; lysine and tryptophan and thus fully
UMYU Journal of Microbiology Research
Their Proteins contain relatively low amounts
of the Sulphur containing amino- acids
methionine and cystine, which are present in
relatively high amounts in the protein of
cereals (Ukonmah and Eruotor, 2012).
Fungi are those that grow on products in
storage; one characteristic that they share in
common is the ability to grow without free
water they comprise several species of
Aspergillus spp. and a few of Penicillium spp.
(Olusegun and Hussaini, 2013). All these have
the ability to grow in grain and legumes. They
occur almost everywhere and contaminate all
grains and legumes (Olusegun and Hussaini,
2013).
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 UJMR, Volume 6 Number 1, June, 2021, pp 208 - 218
Fungi belonging to facultative saprophytes and
facultative parasites may lower the quality of
seeds by causing discoloration, others are;
reduction or elimination of germination
capacity and several other physiological
alterations in grains (Neegaard, 1997).
Aflatoxins are group of secondary fungal
metabolites produced by Aspergillus species,
such as A. flavus and A. parasiticus, A.
bombycis, A. ochraceoroseus, A. nomius, and
A. pseudotamariare also aflatoxin-producing
species, but they are encountered much less
frequently (Bennett and Klich, 2003).
Aflatoxins are potent toxic, carcinogenic,
mutagenic, immunosuppressive and teratogenic
agents produced as secondary metabolites by
A. flavus and A. parasiticus (Krishnamurthy and
Shashikala, 2006).
Aflatoxins become more prevalent, and
therefore more of a food safety concern, during
drought because low rainfall and high
temperatures encourage the growth and
survival of the moulds that produce the toxins.
Crops stressed by drought and high
temperatures and/or weakened by insect or
other damage, are more susceptible to mould
growth and subsequent aflatoxin
contamination. The aflatoxin producing moulds
can grow on crops in the field, poorly dried
harvested crops in storage, processed food, and
feed products (Abbas, 2005).
The production of aflatoxin is enhanced by
environmental conditions (temperature and
relative humidity) and storage conditions.
Other factors include water activity, moisture
content in foods and substrates, in addition to
the damage caused by insects (Arruset al.,
2005).
The major types of aflatoxin are B1, B2, G1, G2,
M1 and M2 (Wrather, 2008). Aflatoxin B1 is
produced most abundantly and the most toxic
followed by G1, B2 and G2 respectively.
Aflatoxin B1, B2, G1 and G2 are classified as
Group I human carcinogens whereas M1 is
classified as Group 2B probable human
carcinogen (Krishnamurthy and Shashikala,
2006). Aflatoxin B1 is responsible for
carcinomas in animals, showing a strong
relationship with the incidence of cancer in
humans (Jolly et al., 2009; Meggs, 2009).
Aflatoxin B1 occurs in the highest amounts in
contaminated commodities; and total aflatoxins
(AFT) refers to the sum of the related
compounds of aflatoxins. According to Food
and Agriculture Organization (FAO), the world
wide maximum tolerant levels of aflatoxin B1
was reported to be in the range of 1–20 µg/kg
in foods, and 5–50 µg/kg in dietary cattle feed
in (FAO, 2004). Aflatoxin B1 is mostly found in
contaminated food and humans are exposed to
aflatoxin B1 almost entirely through their diet.
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Occupational exposure to aflatoxin B1 has also
been reported in swine and poultry production
(Viegaset al., 2013).
This research aimed at determining aflatoxin
contamination in cereals and legumes marketed
in Zaria Metropolis with the view to suggest
control measures and to avoid eating food
contaminated with aflatoxins.
MATERIALS AND METHODS
Study Area
Zaria metropolis is located at latitude 11° 07’ N
and longitude 07°42’ E, and is one of the most
important cities in Northern Nigeria (Ubaet al.,
2008). It has a total area of 300 km2 and
consisting of six major settlements; Zaria City,
Tudun Wada, Sabon Gari, Danmagaji, Kwangila
and Samaru. Zaria metropolis, Kaduna State, is
reported to have had a population of 1,018,827
in 2007 (TWG, 2007).
It has a tropical continental climate with a
pronounced dry season, lasting up to six months
(May - October). During the dry season, a cool
period is usually experienced between
November and February. This emanates from
the influence of the Northeastern winds
(harmattan) which control the tropical
continental air mass coming from the Sahara.
Hazy to dusty conditions and low temperatures
characterize the North-East (NE) winds, as low
as 10°C at night. A temperature of 40°C is
sometimes recorded in the afternoon. The
humidity also drops to less than 15% in
December/January. Zaria experiences a brief
period of hot but dry weather in March and
April, followed by a progressive incursion of
tropical maritime air mass from the Atlantic
Ocean, which displaces the NE (harmattan)
winds. During this short period, the mean daily
maximum temperatures are stable, and they
ranged from 38 - 42°C. After this, the South
Westerly Monsoon winds laden with moisture
bring the rain in thunderstorms and squalls with
heavy fall of high intensities. The rainy season
lasts from May to September/October with
long-term annual rainfall of 1,040 mm in about
90 rain days. The relatively deep tropical
ferruginous soils and climate conditions of Zaria
are suitable and sustain a good cover of
savanna woodland (Northern Guinea Savanna),
with a variety of grasses woody shrubs and
short trees. Six big markets were selected from
the metropolis for analysis.
Collection of Samples
Ninety samples of millet, sorghum, maize,
beans and groundnuts were collected from
major grains sellers from markets in Zaria
metropolis namely Zaria city, Tudun Wada,
Sabon Gari, Kwangila, Danmagaji and Samaru
market in Zaria, from July - August, 2019.
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 UJMR, Volume 6 Number 1, June, 2021, pp 208 - 218
Three vendors were selected at different point
in each market and five samples were
purchased from each vendor. Ninety samples
were collected; 18 samples of millet, sorghum,
maize, beans and groundnuts respectively.
Approximately 100g samples were purchased
from each vendor and the samples were placed
separately in clean, sterile containers labelled an air oven at 105oC, cooled in a desiccator and
appropriately and transported immediately to
the Laboratory for analyses. Each sample was
divided into two; one-half was used for
proximate analysis and other half for
mycological investigation and quantification of
aflatoxins.
% Moisture content = W3 - W1 x 100
W2 - W1
Determination of Ash Content
The porcelain crucible was dried in an oven at
100oC for 10 min, cooled in a desiccator and
weighed (W1). Two grams of the sample was
placed into the previously weighed porcelain
crucible and weighed (W2). The sample was
ignited and transferred into a furnace, which
% Ash content = W3 - W1 x 100
W2 - W1
Determination of Crude Lipid
A clean, dry 500ml round bottom flask,
containing few anti-bumping granules was
weighed (W1) and 300ml of petroleum ether
(40-60oC) for extraction was poured into the
flask fitted with soxhlet extraction unit. The
extractor thimble containing twenty grams of
the sample was fixed into the soxhlet
extraction unit. The round bottom flask and a
condenser were connected to the soxhlet
% Crude Lipid = W2 - W1 x 100
Weight of sample
Determination of Crude Fibre
Two grams of sample was weighed into a round
bottom flask. Hundred millilitres of 0.25M
sulphuric acid solution was added and the
mixture boiled under reflux for 30min. The hot
solution was quickly filtered under sunction.
The insoluble matter was washed several times
with hot water until it was acid free. It was
quantitatively transferred into the flask and
100ml of hot 0.31M sodium hydroxide solution
The loss of weight on incineration =
Determination of Nitrogen Content and Crude
Protein
About 1.5g of the defatted sample in an ash
less filter paper was dropped into 300ml
Kjeldahl flask in order to digest the protein.
Twenty-five millilitres of H2SO4 and 3g of mixed
UMYU Journal of Microbiology Research
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Proximate Compositions of Legumes and cereal
grains
The analyses of proximate composition of the
samples was carried out according to AOAC,
(2010) method.
Determination of Moisture Content
A clean crucible was dried to constant weight in
weighed (W1). Two grams of the sample was
accurately weighed into the previously labelled
crucible and reweighed (W2).
The crucible and sample were dried in oven to
a constant weight (W3). The percentage
moisture content was calculated thus:
was then set at 550oC. The sample was left in
the furnace for eight hours to ensure proper
ashing. The crucible containing the ash was
then removed cooled in the desiccator and
weighed W3. The percentage ash content was
calculated as:
extractor and cold-water circulation was put
on. The heating mantle was switched on and
the heating rate was adjusted until the solvent
was refluxed at a steady rate. Extraction was
carried out for six hours. The solvent was
recovered and the oil dried in the oven at
70oCfor one hour. The round bottom flask
containing the oil was cooled in the desiccator
and then weighed (W2). The lipid content was
calculated thus:
added and the mixture boiled again under
reflux for 30 minutes and quickly filtered under
sunction. The insoluble residue was washed
with boiling water until it was based free. It
was dried to constant weight in the oven at
100oC, cooled in a dessicator and weighed (C1).
It was then incinerated in a muffle furnace at
550oC for 2 hours, cooled in the dessicator and
reweighed (C2).
The crude fibre was calculated thus:
C1 - C2 x 100
Weight of original sample
catalyst (weighed separately into an ash less
filter paper) were dropped into the Kjeldahl
flask. The flask was then transferred to the
Kjeldahl digestion apparatus. The sample was
digested until a clear green colour was
obtained.
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 UJMR, Volume 6 Number 1, June, 2021, pp 208 - 218
The digest was cooled and diluted to 100ml
with distilled water.
Twenty millilitres of the diluted digest were
measured into a 500ml Kjeldahl flask containing
anti-bumping chips and 40ml of 40% NaOH, was
slowly added by the side of the flask. The
250ml conical flask containing a mixture of
50ml of 2% Boric acid and 4 drops of mixed
indicator was used to trap the ammonia
liberated. The conical flask and the Kjeldahl
flask were then placed on the Kjedahl
distillation apparatus, with the tubes inserted
into the conical flask and the Kjedahl flask. The
flask was heated to distil out NH3 evolved. The
distillate was collected into the boric acid
solution. After the boric acid turned green, 10
minutes were allowed for complete distillation
of the ammonia present in the digest. The
distillate was titrated with 0.1M HCl.
The nitrogen content and crude protein were
thus calculated as:
Percentage Nitrogen = 14 x M x Vt x Tv x 100/
Weight of Sample (mg) x Va
% Crude protein = % Nitrogen (N2) x 6.25
Where M = Actual molarity of acid
Tv = Titre volume of HCl used
Vt = Total volume of diluted digest
Va = Aliquot volume distilled
Determination of Carbohydrate Content (by
difference)
The sum of moisture, ash, crude lipid, crude
protein and crude fibre was subtracted from
100% as illustrated below:
% Total Carbohydrate= 100 – (% Moisture + % Ash
+ % Fat + % Protein + % Fibre).
Isolation of Fungal Species
Media Preparation
Sweet potato yeast extract (SPYE) Agar was
compounded as follows; 250g of fresh sweet
potato was cut into smaller pieces and boiled in
1000mL of distilled water for 30 minutes. The
extract was collected in a clean Erlenmeyer
flask by passing the suspension through a clean
muslin cloth. To the total volume of the extract
in the Erlenmeyer flask, 1.3g of yeast extract
and 20g of agar-agar was added and the pH
adjusted to 5.5 and boiled until the agar
dissolved. The contents of the flask were
sterilized in an autoclave at 121oC for
15minutes. After cooling, streptomycin added
to inhibit bacteria growth, poured in sterile
Petri-dishes, and allowed to set and the plates
were dried in an incubator.
Slants of the SPYE agar was also prepared as
described above but in test tubes.
Neutral Red Desiccated Coconut Agar (NRDCA)
was prepared by modification of the method of
Davis et al.,(1987) as reported by Atanda et
al., (2011) as follows; two hundred grams of
desiccated coconut was soaked in hot distilled
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water for 30 min., blended aseptically in a
Warring blender for 5 min and filtered through
two layers of cheese cloth. Two percent agar
(Oxoid) was added to the filtrate, heated to
boiling, cooled to about 50oC. To the filtrate,
0.1% neutral red stain was added and the pH
was adjusted to 4.5. The media was then
sterilized at 121oC for 15 min, cooled and
poured uniformly into sterile Petri dishes.
Inoculation of Samples
Ten gram (10g) of each ground sample of
millet, sorghum, maize, beans and groundnuts
was separately added to 90ml of sterile
distilled water and homogenized for 2 minutes
to form a stock suspension. An aliquot of 0.5ml
was spread on already prepared sweet potato
yeast extract agar plate and the inoculated
plates were incubated at room temperature for
3- 5 days (Bankole and Mabejoke, 2004).
Observed colonies were sub cultured on SPYE
agar in order to obtain pure isolates and the
observed colonies were identified based on
macroscopic and microscopic features (Pitt and
Hocking 2009) and the isolates was sub-cultured
into fresh sweet potato yeast extract agar slant
and kept in the refrigeratoruntilrequired.
Screening of Isolates for Aflatoxigenic
potential
The pure isolates of the samples were screened
for aflatoxigenicity using desiccated neutral red
coconut agar as described by Ezekiel et al.,
(2013). Each isolate was inoculated on freshly
prepared neutral red desiccated coconut agar,
incubated at room temperature for 3-5 days.
Isolates that absorbed and emitted very bright,
moderate and weak UV light (fluorescence) at
365nm were confirmed to be capable of
producing aflatoxins.
Quantification of Aflatoxins by ELISA Method
Sample Preparation and Extraction
Representative samples of millet, sorghum,
maize, beans and groundnuts were pulverized
to about 75% of the sample will pass through a
mesh sieve. Fifty grams of the grounded
samples was collected into a conical flask and
5.0 g of NaCl added. The samples were further
mixed with 100ml of 80% methanol and blended
at high speed for 3 minutes. The samples were
allowed to settle and filtered through filter
paper filtrate was collected and 5ml of the
filtrate was mixed thoroughly with 20ml of
distilled water and filtered through a glass fibre
filter (Beacon, 2015).
Assay Procedure
Ninety-Five wells were placed in a micro well
strip holder one for each sample and the
standards, then 50 micro litres of enzyme
conjugate were measured from the green
capped bottle and dispensed in each test wells.
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 UJMR, Volume 6 Number 1, June, 2021, pp 208 - 218
The micro pipette was used to take 50 micro
litres of each sample and the standard added
into appropriate test wells containing 50 micro
litres of the enzymes conjugate, and then 50
micro litres of antibody was dispensed into
each test well, the plate was shaken gently to
mix the content and incubated at room
temperature for 10 minutes. The contents of
the wells were dumped, the wells were washed
by filling with distilled water by pouring and
dumping it five times carefully in order not
disrupt the wells from the holder during
washing procedure. Following the last wash,
the absorbent paper towel was placed on the
flat surface of the test wells and tapped to
remove the last of the wash solution. Hundred
micro litres of the substrate from blue-capped
bottle was measured and dispensed into each
test wells, the plate was shaken gently and
incubated at room temperature (37oC) for 10
minutes. Hundred micro litres of stop solution
from red capped bottle was measured and
dispensed into each test well and shaken the
plate rack gently. The colour changes from blue
to yellow and then the test wells on micro well
ELISA reader at 450 nm and a differential filter
of 630nm. The optical density (OD) was taken
from each micro well and the concentrations
were obtained from a graph curve that was
obtained from OD and the concentration of the
standards (Beacon, 2015).
Data Analysis
The mean aflatoxin concentration and
proximate compositions of the food
commodities were analysed by Analysis of
Variance (ANOVA) using SPSS Version 20. P
≤0.05 was considered significant.
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and the least in crude fibre (1.92%), crude
protein (5.11%) and crude lipids (4.53%). In
addition, groundnuts samples recorded the
highest concentration of crude lipids (32.36%),
crude protein (16.71%) and crude fibre (4.04%)
and the least concentration of carbohydrates
(36.05%). There were significant differences (P<
0.05) in the Ash, crude lipid, crude protein and
carbohydrate content of the samples (Table 1).
Distribution of Aflatoxigenic Moulds in the
Grain Samples
Forty-eight 48 (53.3%) of the 90 samples were
contaminated with fungal species. The
occurrence of Aspergillus flavus was 31(34.4 %)
while Aspergillus parasiticus was 17(18.9 %).
Millet however had the highest occurrence of A.
flavus 9(50%) and A. Parasiticus 5(28%) while
sorghum has the least occurrence of A.flavus
3(16%) and A.parasiticus 2(11%). Similarly,
maize and groundnut samples had the same
percentage occurrences of 39% and 22%
respectively for A. flavus and A.parasiticus
(Table 2).
Aflatoxin Producing Ability of A. flavus and A.
parasiticus
The two aflatoxigenic moulds (Table 2) were
isolated namely A. flavus (31) and A.
parasiticus (17). Of the A. flavus and A.
parasiticus isolates tested for aflatoxin
production, 07 A. flavus isolates had very bright
fluorescence intensity, while A. parasiticus had
5 withvery bright fluorescence intensity. Nine
isolates of A.flavus had weak fluorescence
intensity followed by A. parasiticus with only 3.
Thus, indicating the ability of the isolates to
produce aflatoxin in large or small quantities
(Figures 1 and 2).

RESULTS
Proximate Composition of the Grains and
Legumes
Sorghum had the highest concentration of
carbohydrates (77.73%) and moisture (9.73%)

Table1: Proximate Composition ofCereal Grains and Legumes (%) Sold in Zaria Metropolis
Food Moisture Ash Crude Crude Crude Carbohydrate
Commodity lipid protein fibre

Millet 8.16a 1.43b 13.40b 5.28b 3.49a 68.30a

a b c b a
Sorghum 9.73 0.98 4.53 5.11 1.92 77.73a
a b c b a
Maize 8.89 0.91 6.03 5.42 3.41 75.23a
a a b b a
Beans 8.38 3.57 17.13 7.18 2.89 60.96a
a a a a a
Groundnut 8.68 2.16 32.36 16.71 4.04 36.05b
Means with different superscript along the columns are significant (P < 0.05)

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Table 2: Distribution of Moulds in Cereal Grains and Legumes Sold in Zaria Metropolis
Food No. of Sample No. of fungal species & % occurrence of Total No. of sample
Commodity Analysed A. flavus A. Parasiticus contaminated
Millet 18 9(50) 5(28) 14
Sorghum 18 3(16) 2(11) 5
Maize 18 7(39) 4(22) 11
Beans 18 5(28) 2(11) 7
Groundnut 18 7(39) 4(22) 11
Total 90 31(34.4) 17(18.9) 48

4
Number of isolates

3 33 33

2 High Ability
Moderate Ability

1 1 1 1 1
Weak Ability

0 0

Millet Maize Groundnut

Source of Fungal Isolates

Figure 1: Aflatoxins Production Ability of A. flavus on Neutral Red Desiccated Coconut Agar
under UV Light (365nm)
3 3 3
Number of Isolates

High Ability
11 11 1 11 1 Moderate Ability
Weak Ability

0 0 0 0

Millet Maize Groundnut

Source of Fungal Isolates

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 UJMR, Volume 6 Number 1, June, 2021, pp 208 - 218 ISSN: 2616 - 0668
Figure 2: Aflatoxigenic Production Ability of A. parasiticus Isolates on Neutral Red Desiccated
Coconut Agar under UV light (365 nm)
Aflatoxin Concentration of Grains and The level of aflatoxins in the samples from Dan-
Legumes Magaji Market was found to be the least with a
The aflatoxin concentration of the grains and concentration of 1.32µg/kg, the highest
legumes marketed in Zaria metropolis, showed concentration of the aflatoxin was found in
that the concentration ranged between 1.07 grain samples from Sabon Gari Market with a
µg/kg (sorghum) to11.04 µg/kg for millet mean concentration of 5.17µg/kg as shown in
(Table 3). There were significant differences (P Table 4 and the samples were not statistically
≤ 0.05) in the level of aflatoxin concentration significant.
of the samples.

Table 3: Mean Aflatoxin Concentrations of Cereals and Grain Legumes Sold in Zaria Metropolis

Food Number of sample Concentration of Aflatoxin

Commodity Analysed Positive Range (µg/kg) Mean*(µg/kg)

Millet 18 9 0.4 – 52.0 11.04a

Sorghum 18 3 3.6 – 11.4 1.07b

Maize 18 7 0.4 – 18.1 2.17b

Beans 18 5 0.5 – 14.7 1.99b

Groundnut 18 7 0.6 – 16.9 2.44b

Means with different superscript along the column are statistically significantly different (p ≤ 0.05).

Table 4: Aflatoxin Concentrations of Cereal Grains and Legumes Marketed in Zaria Metropolis
According to Markets

Market Number of sample Concentration of Aflatoxin

Analysed Positive Range (µg/kg) Mean*(µg/kg)
Zaria City 15 4 2.6 – 52.0 4.45a

Tudun Wada 15 5 2.3 – 9.2 1.89b

Sabon Gari 15 8 0.5 – 29.7 5.17a

Kwangila 15 5 3.0 – 40.0 4.77a

Samaru 15 4 0.6 -39.6 4.87a

Dan-Magaji 15 5 0.4 – 8.0 1.32b

Means with different superscript along the column are statistically significantly different (p ≤ 0.05).

DISCUSSION The percentage ash content is an indication of

The moisture contents observed in the samples
were low. However, long-term storage of the
grains may increase the fungal growth. The
moisture contents observed in this study was
similar to that of Shituet al., 2018that reported
moisture content of between 10.4 –12.7% in
maize, sorghum and millet.Moisture and
temperature are the main factors that
influence post-harvest contamination of stored
commodities by aflatoxigenic moulds (Hell and
Mutegi, 2011).
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minerals in the samples. The sorghum and
maize samples had lesser mineral content
based on their ash contents, whereas the
millets, groundnuts and beans were rich in
minerals required for growth of the moulds.
This observed variation in the ash contents in
the different grains might be due to genetic
factors and environmental factors like irrigation
frequency, soil composition and fertilizers
(Ikram et al., 2010). This is similar with the
1.4-3.3% ash content of maize as reported by
Shituet al., (2018). The ash content observed is
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 UJMR, Volume 6 Number 1, June, 2021, pp 208 - 218
however lower than the 5.1% in maize reported
by Mlayet al. (2005). This might be due to
The percentage crude protein content of the
grains indicated that they were good sources of
nitrogen, which is required for growth of
moulds and aflatoxin production. The crude
protein content observed in this study was
lower than the 20.8 – 23.7% reported by
Habibullah and Hamid, (2007) in beans. The
possible reasons for the variation might be due
to differences in storage conditions in the study
area and genetic factor.
The carbohydrate content of grains indicates
fermentable sugars required for the growth of
moulds and subsequent aflatoxin production.
The range carbohydrate content of the grains is
similar to the 65 – 75%, and 71.7% reported by
Subramanium and Metta (2000) for maize in
India. Isma’il, (2016) however reported a
concentration of 61.37% for sorghum and
Adebolu (2005) reported a concentration of
65.63% for maize. The concentration of beans
was as low as 36.05%. The reasons for the
observed differences could bedue to
lowcarbohydrate constituent of the two major
components of starch (amylose and
amylopectin) during germination or possible
degradation along processing line.
The distribution of moulds isolate in this study
showed that Aspergillus flavus was the most
dominant than the A. parasiticus in all the five
different grains samples analyzed. Millet
samples recorded highest occurrence of A.
flavus and A. parasiticus and produced the
highest aflatoxin concentration than other
samples analyzed, while sorghum samples had
the least occurrence, hence recorded the least
aflatoxin concentration. The observed variation
might be due to differences in storage
practices. The presence of A. flavus and A.
parasiticus however, call for concern, as these
moulds are known to produce aflatoxins and
have been implicated in mycoses.
Between the two species, A. flavus was found
to be dominant mould, which is in agreement
with Klichet al. (2009) who reported a high
incidence of A. flavus. Previous studies had
shown that A. flavus frequently occurred in the
field. The presence of both Aspergillus flavus
and A. parasiticus in the grains and legumes is
very common. According to Jeleneet al., (2013)
droughtcondition caused higher incidence and
favours Aspergillus flavus. Similarly, Rossettoet
al., (2005) also attributed the high frequency of
the two moulds to the adaptation of these fungi
to the substrates, especially during storage.
The high frequency of A. flavus observed in
millet samples may be because of the contact
of the substrate with the soil. The incidence of
the aflatoxigenic moulds in the agricultural
products as observed in this study may be due
UMYU Journal of Microbiology Research
ISSN: 2616 - 0668
agronomic practices like field drying and other
environmental factors.
to inadequate storage and handling practices,
the ubiquitous nature of these moulds and
environmental factors (Ibehet al., 1991).
The production of yellow pigmentation on the
reverse side of plates of neutral red desiccated
coconut agar is an indication of the presence of
aflatoxin producing species thus obviating the
need for UV light in the screening process
(Atandaet al. 2006). Furthermore, Abbas et al.
(2005) suggested the use of fluorescence
production as an effective cultural method for
the detection of aflatoxin producing ability.
The isolates of A. flavus and A. parasiticus
isolated from the samples were found to be
capable of producing aflatoxins using Neutral
Red Desiccated Coconut Agar (NRDCA) under UV
light (365nm). Dyer and McCammon (1994)
reported that the colour fluorescence under UV
light is a useful tool in differentiation of
toxigenic isolates, as A. flavus fluoresced
pastel blue in a ring around each colony, while
A. parasiticus fluoresced bluish white. Thus,
fluorescence colouration could be used in
differentiating A. flavus and A. parasiticus.
This is an indication that neutral red desiccated
coconut agar (NRDCA) enhance the aflatoxin
detection ability of the medium. This finding is
in agreement with the findings of Cotty (1997)
and Yu et al. (2004) who reported that
Aspergillus species produce aflatoxin in
appreciable amounts on DCA by the isolates as
reported by Olsen et al. (2008).
Aflatoxin is an important naturally occurring
mycotoxin in agricultural products. They are
produced by several species of Aspergillus. Not
all strains of Aspergillus species produce
aflatoxins (Frisvadet al., 2007).
Millet is a major staple food in Northern
Nigeria. In this study, the mean level of
aflatoxin found in millet from Sabon Gari
market had the highest aflatoxin contamination
level and concentration range far beyond the
acceptable limit of 4µg/kg set by SON. This
could be due to differences in storage
conditions between the markets. This finding is
not in agreement with that of Batagarawaet al.
(2005) who reported a concentration 0.62µg/kg
in millet from Katsina State and Ezekiel (2014)
who reported a range of 0.08-1.40 µg/kg for
millet. The differences in contamination level
might be due to the difference in
environmental factors (temperature and
relative humidity) that favours the growth of
aflatoxigenic moulds and agricultural practices
between the two study areas.
Sorghum is another staple food and an
important starchy food for human and animal
consumption in Nigeria. The mean level
aflatoxins found in the sorghum samples are
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 UJMR, Volume 6 Number 1, June, 2021, pp 208 - 218
within the acceptable limit. However, the
range of aflatoxin concentration of sorghum
samples from Kwangila market exceeded the
limit set standard by NAFDAC, thus others are
safe for human consumption. The low level of
aflatoxin observed in sorghum might be due to
the high phenol and tannin contents present in
sorghum, which are known to inhibit fungal
infestation (U.S. Grain Council, 2008). Though
the aflatoxin concentration levels in samples
were not disquieting, consistent consumption
might result in long-term accumulation of the
toxins, causing disease and metabolic disorder
resulting in poor human and animal health.
The differences observed in aflatoxins
concentration between millet and sorghum
could be due to environmental factor
(temperature and humidity), storage condition,
handling processing and sensitivity of the
method of quantifying the aflatoxins.
Maize is one of the most widely distributed
food plants in the world (Bradburn, 1993).
Maize samples analyzed in this study revealed
that the mean aflatoxin concentration and the
range are within the acceptable limit, except
maize sample from Samaru market that had the
highest level of contamination, which exceeded
the limits sets by SON. The observed
differences within the markets might be due to
prolonged storage and method of handling.
Atehnkenget al. (2008) had earlier reported a
concentration range of between 30.9 µg/kg-
507.9µg/kg for maize from 11 districts across
three agro-ecological zones of Nigeria. Manjula
(2009) also reported mean aflatoxin levels of
0.55µg/kg and 0.46µg/kg in maize samples.
The contamination levels of groundnuts were
within the acceptable limit except for samples
obtained from Sabon Gari market, which
exceeded the acceptable limit and is call for
concern. This could also be due to handling
practices, storage conditions and other
environmental factors. This finding differed
from that of Batagarawaet al. (2015) where
6.68µg/kg was for reported in groundnut and
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